Figure 1. E. carinatus venom stimulates ex vivo NETosis.

E. carinatus venom-stimulated NET formation was quantified using (a) MPO-DNA capture ELISA and (b) Hoechst staining in dose- (left) and time-dependent (right) assays. The results are expressed as the percent increase relative to unstimulated cells (US); mean±s.e.m. (n=6). *P<0.05, **P<0.01, ***P<0.001 versus US; one-way analysis of variance (ANOVA), followed by Dunnett's post-hoc test. PMA (50 nM) served as a positive control. (c) Western blot analysis of PAD4 expression (top, left) and the presence of H3Cit (top, right) in E. carinatus venom-treated neutrophils. PAD4 expression was normalized to GAPDH expression (bottom, left), and H3Cit levels were normalized to H3 levels (bottom, right). AU, arbitrary units; H3Cit, citrullinated histone 3; H3, histone 3; US, unstimulated cells. The data are presented as mean±s.e.m. (n=4). ***P<0.001 versus US; one-way ANOVA, followed by Dunnett's post-hoc test. PMA (50 nM) served as a positive control. The PVDF membranes were cut based on molecular weight of respective protein using protein molecular weight marker and then probed with respective antibodies. (d) Representative immunofluorescence images of neutrophils/NETs. The neutrophils were exposed to E. carinatus venom (25 μg ml⁻¹) for 2.5 h at 37 °C. Yellow arrows indicate NETs. (n=4) Scale bars, 100 μm. PMA (50 nM) served as a positive control. (e) Scanning electron microscopy images showing unstimulated neutrophils (left) and E. carinatus venom-stimulated neutrophils, which displayed NETs with thick bundles of fibres (black arrowheads; right). (n=4) Scale bars, 30 μm.